Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.     

Involvement of arabinogalactan proteins in the regeneration process of cultured protoplasts of Marchantia polymorpha

Protoplasts of Marchantia polymorpha L. (liverwort) regenerated new cell walls in initial culture. However, the survival rate of regenerated cells decreased rapidly after this stage. The decrease in survival rate was suppressed by the β-glucosyl Yariv reagent (βglcY), which binds to arabinogalactan proteins (AGPs), only when it was added to culture medium during the period of incipient cell wall regeneration. The addition of βglcY after the period of incipient cell wall regeneration had no effect on the survival rate. These results suggested the involvement of AGPs in the cell wall regeneration process. After cell wall regeneration, the regenerated cells started to divide actively after being transferred to a medium with 1% activated charcoal (AC). Protoplasts that had been cultured with βglcY during the period of incipient cell wall regeneration and then transferred to the AC medium divided vigorously, and the cell division rate was remarkably increased (>80%). However, without transfer to the AC medium, βglcY at concentrations higher than 20 μg ml⁻¹ inhibited cell division. No effect on cell survival nor cell division was observed with the α-galactosyl Yariv reagent. Staining of β-1,3-glucan (callose) with aniline blue (AB) showed that a large amount of β-1,3-glucan was deposited in the regenerated cell walls of the protoplasts cultured without βglcY, while little or no β-1,3-glucan was stained by AB in protoplasts cultured with βglcY. These results suggest that AGPs and β-1,3-glucan play important roles in the survival and subsequent cell division of regenerated cells of M. polymorpha protoplast cultures.     

Plant Regeneration from Protoplasts of Actinidia chinensis Planch.

Protoplasts isolated from cotyledon callus line of A14N7 of Actinidia Chinensis Planch. were cultured in the improved NN-69 medium. First division of regenerated cells occurred during 7–10 days of culture, and percentage of the cell division was about 10% at day 20. The best result of protoplast culture was achieved when protoplasts were cukured in liquid medium at a density of 5× 104/ml, About 4 months, procoplast-derived calli were transferred stepwisely onto differentiation media where they developed into green compact calli, from which the perfect plants were regenerated.     

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10⁴ protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

Haploid callus and regeneration of plants from anthers of Digitalis purpurea L.

Summary Production of callus from anthers of D. purpurea was obtained on several basal media supplemented with various amounts of auxins. Chromosome counts showed that the callus produced was haploid when the anthers 1) were of a dark-brown to black color, and 2) were cultured in the late tetrad stage of microspore development. Subsequent differentiation to plants at high frequencies was possible only 1) when the anthers had been cultured on the medium of Nitsch and Nitsch (Science 163, 85–87; 1969) supplemented with 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2) when the callus was transferred to the same medium but without 2,4-D, and 3) when it was cultured under continuous light from fluorescent lamps. Proliferation of the callus and regeneration of plants did not diminish through as many as 20 subcultures. The high frequency of regenerates permits the propagation of a distinct geno-type to a virtually unlimited number of plants. Diploid plants were obtained when the anthers had been cultured in the dark. Tetraploid plants were regenerated by callus from anthers which had been cultured in light. When the time of 2,4-D treatment was shortened a few haploid plants were produced which however did not survive transfer to soil. Cytological observations demonstrated that regeneration started from haploid callus, leading to intermediate degrees of ploidy and finally to diploid plants. Most of the regenerated plants were euploid and flowered and fruited normally under greenhouse and field conditions. If the anther-derived callus was cultured on the medium of Nitsch and Nitsch supplemented with 2.2 mg/l kinetin, plants regenerated only under photoperiodic conditions of 16 h light at 28° and 8 h dark at 20° but the survival was lowered to one third. These plants had a different leaf and flower morphology as compared to the control without kinetin and to the starting material, but their progeny was again essentially normal.     

In vitro plant regeneration from cotyledon fragments of the olive tree (Olea europaea L)

Olive tree (Olea europaea L) plantlets were regenerated from cotyledon segment calli on a modified olive medium (OMc) supplemented with 2iP alone or in combination with indol-3-butyric acid (IBA). Cell division in the explants was initially induced on OMc medium with high auxin (5 mg·l⁻¹ of IBA) and low cytokinin (0.2–0.5 mg·l⁻¹ of 2-isopentenyladenine (2iP) or zeatin riboside) content. Calli were then transferred to the same medium with different levels of IBA and/or 2iP in order to promote further development and obtain calli bearing either roots or shoots. On OMc medium, 1 mg·l⁻¹ of IBA induced the maximum of rooting, while shoot induction was greater when the medium was supplemented with 4 mg·l⁻¹ of 2iP. Shoot induction mainly occurred from calli of cotyledon fragments proximal to the embryo axes. Whole plantlets were obtained when the regenerated shoots were stimulated to produce adventitious roots on OMr medium with 1 mg·l⁻¹ of IBA or naphthaleneacetic acid (NAA). After root elongation on OMe medium without auxin, plantlets were transfered to peat and soil conditions where about 75–80% were able to survive. A certain variability was detected between regenerated olive plants.     

High Efficiency Plant Regeneration from Protoplasts of the Indica Cultivar IR58

Procedures have been established for embryogenic cell suspension,protoplast culture, and plant regeneration from the Indica ricecultivar, IR58. Embryogenic cell suspension was establishedin modified R2 medium and maintained in amino acid-based AAmedium. Protoplasts were cultured in R2 basal medium containingB5 vitamins. Plants were regenerated in MS basal medium with3% maltose as carbon source. Selection of embryogenic protoplast-derivedcalli is an important step for high-frequency plant regeneration.More than 900 plants were regenerated through this procedure. Key words: Rice (Oryza sativa, Indica, Protoplast, Maltose     

甘蓝下胚轴原生质体再生植株

经纯化后,甘蓝下胚轴原生质体的产量为1．5×10⁶g^-1(Fw),采用液体浅层培养的方法进行培养。2～3d后,发生第一次分裂,第10天,统计分裂频率为6l％,5周内形成大量的细胞团和小愈伤组织,统计植板率为1．1％,把小愈伤组织转到与原生质体培养基相同激素的MS固体培养基上增殖。当愈伤组织长到3～5mm大小时,接到分化培养基上,芽分化率为46.7％．分化出来的芽长到3～4cm长时,从基部切下,插入生根培养基,两星期左右即可长成完整植株。     

Influence of the Antibiotic Ciprofloxacin on Culture of Allium longicuspis Callus-derived Protoplasts

A major problem of in vitro plant culture techniques is chroniccontamination by microorganisms. Calli derived from basal partsof leaves of Allium longicuspis Regel (Alliaceae) and culturedin a medium without antibiotic contain most probably latentcontaminating microorganisms. These calli were used as the sourcematerial for isolation and culture of protoplasts. Isolatedprotoplasts were cultured in the presence of the antibioticciprofloxacin, and the protoplast viability, cell wall regenerationand cell division were studied as a function of the antibioticconcentration. Whatever the antibiotic concentration, protoplast-derivedcells kept significantly higher viability for at least 3 weekscompared with those cultured without antibiotic. As to cellwall regeneration after 2 d, it was not affected by the antibioticexcept at the highest concentration tested (100 mg l^-1). Sporadicfirst cell division was observed after 2-6 d of culture in thepresence of ciprofloxacin while, in its absence, cell divisionwas never apparent before 10 d of culture.Copyright 1995, 1999Academic Press Allium, bacteria, cell division, cell wall regeneration, ciprofloxacin, contamination, garlic, mycoplasma, protoplast culture, viability     

SHORT COMMUNICATION Factors Affecting Protoplast Culture ofCucumis melo'Green Delica'

Hypocotyls, cotyledons and etiolated half-expanded leaves ofCucumismelo‘Green Delica’ were used as explants for protoplastisolation and culture. Protoplasts isolated from cotyledonsand etiolated half-expanded leaves cultured in Durand, Potrykusand Donn (DPD) medium supplemented with 0.9 µMbenzylaminopurine(BAP), 3.6 µM2,4-dichlorophenoxyacetic acid (2,4-D) and1% sucrose, using the agarose bead culture method, were ableto form cell walls and subsequently go through cell division.Pretreatment of half-expanded leaf explants in the dark for14 d provided the best material for protoplast isolation andcell division. Approximately one third of protoplasts from etiolatedhalf-expanded leaves formed microcolonies. For hypocotyl protoplasts,none of the treatments used were suitable to induce cell division.There was no significant difference between sucrose, glucose,and sucrose plus glucose, in culture media on the plating efficiencyof leaf protoplasts ofC. melo‘Green Delica’; however,bigger colonies were formed in media supplemented with 1% sucrose.No shoot or whole plant regeneration was achieved. However,the methods reported here provide further information onC. meloprotoplastculture.Copyright 1998 Annals of Botany Company Cucumis melo,protoplast culture, 2,4-D, BAP, yeast extract, casein hydrolysate.     

The differentiation of L5/A10 myoblast cell line (a subclone of L5 line) is controlled by changes of culture conditions

We report here that it is possible to induce differentiation in a subline of L5 myoblast line (L5/A10) by manipulating the culture media. When L5/A10 myoblast are cultured in F14 supplemented with 10% fetal calf serum the cells grow with a division time of 12 h and reach confluency at a cell density of approximately 2.4 X 10(5) cells per cm2, without undergoing differentiation, characterized, morphologically, by formation of multinucleated fibers, and biochemically, by the synthesis of muscle specific proteins such as creatine phosphokinase or myokinase. However, cells, grown in F14 + 10% fetal calf serum, will undergo regular differentiation after a limited number of division when transferred to F14 medium supplemented with limiting concentrations (1-2%) of fetal calf serum. Investigations of the biochemistry of myoblast differentiation in cell culture will be facilitated by the availability of a cell line that can undergo differentiation under controlled conditions.     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Thidiazuron induced somatic embryogenesis and plant regeneration in Capsicum annuum

An efficient protocol of direct somatic embryogenesis (without involving intermediate callus) has been developed from stem segments and shoot tips of Capsicum annuum L. Explants were cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). Among the various concentration of TDZ tested, 0.5 μM was proved to be best for induction of somatic embryos. Induction, maturation and germination were achieved on the same medium. The shoots developed from somatic embryos were transferred for rooting to MS medium supplemented with indole-3-butyric acid (IBA). All the regenerated plants with 85 % survival rate were normal with respect to morphology and growth characteristics.     

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis

Protoplasts isolated from cell suspension culture of Phalaenopsis “Wataboushi” were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l l-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.     

骆驼刺发根农杆菌转化系的原生质体培养和植株再生

从发根农杆菌A4转化的荒漠植物—璐驼刺毛状根愈伤组织中分离的原生质体培养的结果表明,酶解新转代7～10d的淡黄色松软愈伤组织,可获得大量有活力的原生质体。原生质体在附加有1．5mg．L-1 2,4．D、0．2mg．L-1 6．BA、0．3m01．L-1甘露醇、2％（W/V）蔗糖和500mg·L-1水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂。培养基的最适渗透压为（450±3）mOsm·kg-1,原生质体的最适植板密度为4×10^5个．mL-1。制备原生质体的愈伤组织以低温（4℃）预处理后,原生质体的产率和分裂频率均提高,分裂频率最高可达50％。原生质体分裂形成的愈伤组织转移在附加1-2mg．L-1 6-BA（或KT）和0．2mg·L-1NAA的MS培养基上培养后,可以分化并获得再生植株。纸电泳检测表明,原生质体再生的愈伤组织和分化植株仍然含有毛状根转化系的特异产物——冠瘿碱。     

Plant regeneration from coleoptile tissue of wheat (Triticum aestivum L.)

Plant regeneration was achieved from coleoptile tissue of wheat (Triticum aestivum L. cv. Kharachia-65). Coleoptiles (1.0 - 3.5 cm long) were excised from 2- to 5-d-old seedlings and cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D - 0.5, 2.5, and 5.0 mg dm^-3). Cream, friable callus was obtained after 6 weeks of inoculation. This callus was sub-cultured on MS medium supplemented with 2,4-D (2.5 mg dm^-3) and 5 % coconut water. After 6 weeks of sub-culturing white, cream or pale, friable, nodular callus was obtained. Plant regeneration occurred when this callus was sub-cultured on MS medium supplemented with 0.2 mg dm^-3 1-naphthalene acetic acid + 1.0 mg dm^-3 6-benzylaminopurine. For rooting, regenerated shoots or plantlets were transferred on MS medium supplemented with 0.5 mg dm^-3 indole-3-acetic acid. Rooted plantlets were directly transferred into pots and grown under field conditions. Seed setting invariably occurred in all plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation,Culture and Callus Formation of lpomoea batatas Protoplasts

Numerous viable protoplasts from stem callus cells of Ipomoea batatas tissue culture have been isolated by enzyme treatment involving cellulase EA3 867 (2.0%), CaC12·2H2O (20 mM) and mineral constituent of medium A at pH5.4 in 0.8 M mannitol in 5 hours at 25±1℃. The protoplasts were cultured at a density of 1-2 × 105/ml in solid agar medium E supplemented with 2, 4-D (0.1mg/l) and kinetin (0.1 mg/l), or NAA (0.3 mg/l) and kinetin (0.1 mg/l) in petri dishes, and placed in a controlled growth cabinet maintained at 27 ℃, and illuminated with floureseent light. They regenerated new cell wails after 7 days of culture. The first cell division was observed after 10 days. Ceil division continued thereafter, and after 40 days of culture small white calli (size about 0.2–0.3 mm) were visible in the petri dishes small calli were inoculated in the same nutrients as the protoplasts culture media, but without mannitol. They developed into large calli.     

埃斯基红豆草下胚轴愈伤组织原生质体的培养与植株再生

埃斯基红豆幼苗的下胚轴切段在附加２,４－Ｄ０．５ｍｇ／Ｌ,ＫＴ１ｍｇ／Ｌ的ＭＳ中形成胚性愈伤组织。来自１１－１３个月龄、继代６－１５天的愈伤组织的原生质体,在改良的Ｖ－ＫＭ液体培养基中可持续分裂形成细胞团,培养１０天时的分裂率和克隆率分别为６５．８８％和５３．３８％周后就可将将原生质体形成的小愈伤组织转于培养基上。原生质体在改良的Ｂ５液体培养基也可以分裂形成小愈伤组织,但分裂率低于Ｖ－ＫＭ。来自原     

Direct Organogenesis and Plant Regeneration in Preconditioned Tissue Cultures of Lathyrus cicera L., L. ochrus (L.)DC. and L. sativus L.

This study demonstrates the importance of preconditioning ofsource tissue in regeneration of multiple shoot buds from severalspecies of Lathyrus. Preconditioned multiple shoots of Lathyruscicera L., L. ochrus (L.) DC. and L. sativus L. were obtainedby germinating seeds on Murashige and Skoog (MS) medium containing50 µM N⁵-benzylaminopurine (BAP) for 2 to 3 weeks. Multipleshoot bud formation occurred when epicotyl explants of preconditionedshoots were cultured on MS medium containing 5–50 µMBAP. No shoot regeneration was observed from epicotyl explantswhich were obtained from non-preconditioned shoots. Shoot budswere formed directly on explants without an intervening callusphase after 2 to 3 weeks of culture. Regenerated shoot budsformed healthy shoots which developed prolific and strong rootswhen transferred to MS medium supplemented with 2.5 µMnaphthaleneacetic acid (NAA). Lathyrus cicera L., L. ochrus (L.) DC., Ochrus Vetch, L. sativus L., Lathyrus pea, de novo differentiation, epicotyl, preconditioning with BAP